This invention relates to the preparation of biologically acceptable bivalent metal coordination complexes. More particularly this invention relates to the hydrolysis of proteinaceous materials under neutral conditions and to the preparation of metal coordination complexes utilizing the protein hydrolysates as ligands.
Metal coordination complexes and more particularly metal proteinates are known in the art to increase the level of bivalent metals in the tissues of both plant and animal organisms.
U.S. Pat. No. 3,873,296 teaches the use of metal proteinates in increasing the levels of essential bivalent metals in plant tissues. A method of increasing bivalent metals in animal tissues utilizing metal proteinates is claimed in copending application Ser. No. 658,243, filed Feb. 11, 1976 and now U.S. Pat. No. 4,020,158.
Metal proteinates are defined as coordination complexes of two or more protein hydrolysate ligands with a metal ion having a valence or oxidation state of at least +2.
There are many products identified in the literature as being reaction products of proteins or protein hydrolysates with metals which are salts or complexes of sorts but most are not metal proteinates.
For example, U.S. Pat. No. 1,824,018 teaches combinations of iron and copper compounds for the treatment of anemia. As related to the present invention, the example showing the combination of copper caseinate and iron peptonate is perhaps most pertinent. Copper caseinate is a combination of copper with casein which has not been hydrolyzed and which may be defined as an insoluble protein salt. This product may be described as the interaction of a metallic copper with an intact protein, but does not produce a metal proteinate or coordination complex as hereinafter described. Likewise, this patent discloses an iron peptonate. Iron peptonate is described in the National Formulary V as a compound of iron oxide and peptone rendered soluble by the presence of sodium citrate. Again, this is not a metal proteinate or coordination complex as will be described.
U.S. Pat. No. 505,985 describes an iron albumen preparation which is a combination of an unhydrolyzed protein which has been heat coagulated. The iron present attaches itself to the surface of the protein molecule and a precipitate is formed. According to the patent this product is readily soluble in a weakly alkaline solution. It cannot therefore be considered to be a metal coordination complex formed from a protein hydrolysate for such a product would be insoluble in basic solution.
U.S. Pat. No. 2,481,413 teaches the preparation of metal caseinates which again are salts or compounds of a protein molecule with metal ions attached thereto, but are not metal coordination complexes.
A more recently issued patent, U.S. Pat. No. 2,960,406 teaches soluble trace metals which are allegedly coordination complexes, preferably using EDTA as a complexing agent. According to that patent, when any of the described metal salts are introduced into water, metal coordination complexes will form in the water solution. While EDTA and its salts are strong complexing agents protein hydrolysates such as naturally occurring amine acids, peptides and polypeptides will not form a coordination complex merely by their combination with a soluble metal salt in water. Therefore, while U.S. Pat. No. 2,960,406 may teach coordination complexes with EDTA and its derivatives, there are insufficient data in the patent to teach the preparation of metal proteinates. The mere combination of a metal salt and a naturally occurring amino acids or protein hydrolysate in an aqueous solution is insufficient for reasons later hereinafter described.
Additionally, U.S. Pat. No. 3,463,858 teaches a process of making a feed additive by slurrying a mixture containing an amino acid source and a water soluble zinc salt, heating, acidifying and drying the slurry. Since this patent teaches that the optimum pH for combining zinc with amino acids is about 3.5, this is inimical to coordination complex formation.
Essential metals such as iron, zinc, copper, magnesium, manganese, calcium, cobalt, molybdenium and chromium are capable of existing in bivalent form. By "bivalent" is meant the metals may assume an ionic or oxidation state of at least +2 or higher.
Since, in general, these metals, as inorganic or organic salts, including those described in the above prior art, are absorbed into the body with difficulty, it is desirable to formulate these metals as proteinates so that they can be effectively assimilated into the body. Salts ionize in the gastric juices of the stomach and enter the small intestine, where most absorption takes place. The intestinal walls are lined with electrical charges which have a strong tendency to repel the positive metal cations while allowing the aninons to pass through. The essential metals are thus discharged through the bowel often causing diarrhea. The anions passing through the intestinal wall often are excreted via the urine and may act as diuretics.
It would therefore be beneficial to prepare a formulation of an essential bivalent metal in a form whereby the effects of the electrical charges of the intestinal lining of the metal were minimized and whereby the metal could readily pass through the acid stomach juices and be made available to the body in a ready assimilable form.
In the past it has been known to utilize certain protein hydrolysates as complexing agents or ligands to increase the assimilation of metals into biological tissues. It has been found however that certain protein hydrolysates are poor ligands due to their size and stereo chemistry. Long chain polypeptides when used as ligands do not form as strong a bond with a metal ion in coordination complex formation as do amino acids, dipeptides and tripeptides. It is therefore axiomatic that coordination complexes formed from metal ions and long chain polypeptides are more easily destroyed in the acidic gastric juices of the stomach.
Protein hydrolysates is a term generally used for any form of hydrolyzed protein ranging from the above mentioned long chain polypeptides down to the basic protein building blocks, i.e. amino acids. These hydrolysates are commonly formed utilizing acidic or basic hydrolysis or a combination of both. Since many different amino acids are essential to the body there is a distinct disadvantage to the utilization of either form of hydrolysis. Acidic hydrolysis destroys the amino acids tryptophan, serine and theonine. On the other hand, basic hydrolysis racemizes the amino acids into their D. and L. forms and destroys arginine, threonine, serine, and cystine. Naturally occurring amino acids belong only to the L-series. Moreover acidic and basic hydrolytic processes require neutralization and this results in the formation of inorganic salts which often remain with and form a material part of the hydrolyzed product.
U.S. Pat. No. 3,396,104 teaches a process for the indiscriminate preparation of metal proteinates utilizing a protein source with saline water and hydrolyzing the protein by a base-acid process to form a proteinate of sorts. The product is not well defined and may vary greatly in mineral content as well as ligand.
An improvement to the above mentioned process is disclosed in U.S. Pat. No. 3,775,132 wherein the protein is subjected to a base-acid-base hydrolysis step and then admixed with a metal salt in alkaline media to form a proteinate.
An enzymatic form of hydrolysis is taught in U.S. Pat. No. 3,857,966 wherein a protein hydrolysate is formed in a two step process wherein a protein source is heat treated under basic conditions and reacted with an alkaline microbial protease and then reacted in a second step at a neutral pH with both a plant enzyme and a neutral microbial protease.
All of the above process are multistep methods requiring at least two different hydrolysis phases.
A milder form of hydrolysis is taught in U.S. Pat. No. 3,969,540 wherein a protein source is enzymatically hydrolyzed under essentially neutral conditions to a polypeptide stage with the hydrolysis being sufficiently mild that no single amino acids are formed. The hydrolysis is not complete and thus the metal proteinate subsequently formed under alkaline conditions contains relatively large amounts of unhydrolyzed or partially hydrolyzed protein such as muscle, heart, liver and the like.